The airway epithelium secretes relaxing factor(s) that may be important in control of airway tone. The chemical identity of these species has not been well characterized. Nitric oxide is a powerful smooth muscle-relaxant implicated in the regulation of blood pressure and gastrointestinal motility. However, the pharmacological literature addressing its role in the airways in general, and as a relaxing factor derived from epithelium, in particular, is controversial. Moreover, one is faced with a major paradox in the lung, namely, that the biological activity of NO needs to be reconciled with its established toxicity in an oxygen-rich environment. We have proposed that answers to paradox lie in the redox chemistry of NO, specifically its capacity to form adducts with both amino acid and protein thiols. These S-nitrosothiols (RSNO) are resistant to reaction with oxygen and possess smooth muscle relaxant activity. The central hypothesis of this proposal is that the epithelium is a rich source of NO, and that reactions with thiol groups play a critical role in its cellular biochemistry and airway pharmacology. The Specific Aims of this project are: 1) to undertake the immunohistochemical, biochemical and molecular characterization of the airway epithelial NO synthase(s); 2) to identify and chemically characterize the endogenous nitrogen oxides and RSNO species synthesized by airway epithelium; 3) to define the biochemical pathways of RSNO formation and metabolism in airway epithelial cells; and 4) to determine the effects of epithelial-derived NO and RSNO on airway function in selected animal models, normal human airways and states of airway hyperreactivity. By demonstrating the physiological importance of NO in airway epithelium, chemically characterizing the adducts formed in epithelial cells and secreted into their environment and defining the bronchodilator activities of endogenous NO species, we expect to identify novel pharmacological approaches for treatment of diseases characterized by airway hyperreactivity.